Differential Expression of Desulfovibrio vulgaris Genes in Response to Cu(II) and Hg(II) Toxicity

Abstract

The response of Desulfovibrio vulgaris to Cu(II) and Hg(II) was characterized. Both metals increased the lag phase, and Cu(II) reduced cell yield at concentrations as low as 50 M. mRNA expression was analyzed using random arbitrarily primed PCR, differential display, and quantitative PCR. Both Cu(II) and Hg(II) (50 M) caused upregulation of mRNA expression for an ATP binding protein (ORF2004) and an ATPase (ORF856) with four-to sixfold increases for Hg(II) and 1.4-to 3-fold increases with Cu(II). These results suggest that D. vulgaris uses an ATP-dependent mechanism for adapting to toxic metals in the environment.Sulfate-reducing bacteria (SRB) are known to be involved in metal bioremediation and for this purpose are often encouraged to grow in engineered systems. They can directly reduce and precipitate many metals, including uranium (11), technetium (9, 10), arsenate (12), and chromium (23), or precipitate metal cations, as metal sulfides (4, 24). Sulfide itself is also known to reduce certain oxidized metals (20), resulting in precipitation. Bioremediation processes may be conducted in bioreactors (4, 24, 25) or in situ (17). While SRB are capable of varied metal transformations, metals can also inhibit their growth (14-16). However, we are not aware of previous research aimed at addressing mechanisms of resistance to divalent metal ions by SRB.Differential display using random arbitrarily primed PCR (RAP-PCR) has been used to identify genes induced by growth under different conditions (1, 2, 8). These techniques were recently used by our group with Desulfovibrio desulfuricans subsp. aestuarii to identify differentially transcribed genes during growth on lactate or hydrogen (22). In the present study, we have used a similar approach to identify genes involved in metal resistance. After first determining metal concentrations needed to inhibit growth of Desulfovibrio vulgaris, we used RAP-PCR to identify genes whose transcription was enhanced in the presence of Cu 2ϩ or Hg 2ϩ . These transcripts were subsequently quantitated in metal-treated cultures and control cultures and were identified through genetic database analysis.Effects of metals on growth. Initial studies were aimed at determining suitable metal concentrations for differential expression experiments. D. vulgaris Hildenborough was grown anaerobically on lactate sulfate medium (LSM) and pyruvate medium (PM) without sulfide, bicarbonate, and CO 2 . LSM was prepared as previously described (7) and contained sodium DLlactate (50 mM), Na 2 SO 4 (50 mM), yeast extract (0.1%), vitamins, minerals, trace metals, and resazurin. D. vulgaris was also cultivated fermentatively in a similar pyruvate medium with pyruvate (20 mM) and MgCl 2 instead of MgSO 4 · 7H 2 O and no other sources of sulfate.For the preparation of inocula, cultures were grown to midexponential phase and harvested by centrifugation (4,650 ϫ g for 20 min). The cell pellet was washed twice and finally resuspended in fresh medium (without bicarbonate-sulfide). Cultures for metal inhibition and e...

Effects of sulfide on sulfate reducing bacteria in response to Cu(II), Hg(II) and Cr(VI) toxicity

“…Upon sulfide addition, the sulfate concentration profiles in the vials containing different Hg(II) concentrations barely changed. This observation is in agreement with the results of Chang et al [16] who reported that Hg(II) increased the lag phase of sulfate reduction but did not affect the SRB growth rate. Compared with Figure 4(c) the lag phase time for each vial in Figure 4(d) decreased from 45 to 25 h and the sulfate reduction rate in each vial also increased.…”

“…Because SRB were identified as the primary organisms responsible for monomethylmercury (MeHg) production during the biotic transformation of inorganic mercury and Hg(II) through energy-dependent uptake systems [16,18,19], the toxic resistance of SRB to Hg(II) was higher than that of other bacteria. At an initial Hg(II) concentration lower than 50 mg L -1 sulfate reduction was not completely inhibited (Figure 4(c)).…”

RNA Arbitrarily Primed PCR and Fourier Transform Infrared Spectroscopy Reveal Plasticity in the Acid Tolerance Response ofStreptococcus macedonicus

“…RAP-PCR was originally adapted from the differential-display PCR method, and it is a powerful technique for the analysis of transcriptional changes, especially those of organisms for which very little or no genetic information is available (6,22). Even though more robust or modern techniques have been developed for gene expression profiling, RAP-PCR has in many cases significant advantages (5,10,36), and it is still being used as a valid method for many studies performed with different organisms (4,17,23,34,37). In our case, we employed this technique to determine transcriptional changes of S. macedonicus due to acid adaptation.…”

Modulation of Medium pH by Caulobacter crescentus Facilitates Recovery from Uranium-Induced Growth Arrest

“…Antimicrobial compounds such as heavy metals and antibiotics are common causes of growth arrest in microorganisms (32,(35)(36)(37)(38). In many cases, recovery from growth arrest is attributed to the induction of a genetically encoded resistance mechanism or the selection of antimicrobial-tolerant mutants during antimicrobial exposure (32,36).…”